Mutagenesis in the alpha3alpha4 GyrA helix and in the Toprim domain of GyrB refines the contribution of Mycobacterium tuberculosis DNA gyrase to intrinsic resistance to quinolones.
ABSTRACT: The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.
Project description:The genes encoding the DNA gyrase A and B subunits of Bacteroides fragilis were cloned and sequenced. The gyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed in Escherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.
Project description:The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.
Project description:The sequence of the DNA gyrase gyrA gene of Serratia marcescens ATCC 14756 was determined. An open reading frame of 2,640 nucleotides coding for a polypeptide with a calculated molecular mass of 97,460 was found, and its sequence complemented the sequence of an Escherichia coli gyrA temperature-sensitive mutation. Analysis of the PCR products of the quinolone resistance-determining regions of gyrA genes from six quinolone-resistant clinical isolates revealed a single amino acid substitution, Ser-83 to Arg or Asp-87 to Tyr, in all six mutants, suggesting that a mutational alteration in gyrA is a common mechanism of quinolone resistance in S. marcescens.
Project description:DNA gyrase mutations are a major cause of quinolone resistance in Mycobacterium tuberculosis We therefore conducted the first comprehensive study to determine the diversity of gyrase mutations in pre-extensively drug-resistant (pre-XDR) (n = 71) and extensively drug-resistant (XDR) (n = 30) Thai clinical tuberculosis (TB) isolates. All pre-XDR-TB and XDR-TB isolates carried at least one mutation within the quinolone resistance-determining region of GyrA (G88A [1.1%], A90V [17.4%], S91P [1.1%], or D94A/G/H/N/V/Y [72.7%]) or GyrB (D533A [1.1%], N538D [1.1%], or E540D [2.2%]). MIC and DNA gyrase supercoiling inhibition assays were performed to determine the role of gyrase mutations in quinolone resistance. Compared to the MICs against M. tuberculosis H37Rv, the levels of resistance to all quinolones tested in the isolates that carried GyrA-D94G or GyrB-N538D (8- to 32-fold increase) were significantly higher than those in isolates bearing GyrA-D94A or GyrA-A90V (2- to 8-fold increase) (P < 0.01). Intriguingly, GyrB-E540D led to a dramatic resistance to later-generation quinolones, including moxifloxacin, gatifloxacin, and sparfloxacin (8- to 16-fold increases in MICs and 8.3- to 11.2-fold increases in 50% inhibitory concentrations [IC50s]). However, GyrB-E540D caused low-level resistance to early-generation quinolones, including ofloxacin, levofloxacin, and ciprofloxacin (2- to 4-fold increases in MICs and 1.5- to 2.0-fold increases in IC50s). In the present study, DC-159a was the most active antituberculosis agent and was little affected by the gyrase mutations described above. Our findings suggest that although they are rare, gyrB mutations have a notable role in quinolone resistance, which may provide clues to the molecular basis of estimating quinolone resistance levels for drug and dose selection.
Project description:Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.
Project description:A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.
Project description:Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg2+-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This "dione resistance-determining region" overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg2+ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg2+ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg2+-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.
Project description:Mutations in gyrA are the primary cause of quinolone resistance encountered in gram-negative clinical isolates. The prospect of this work was to analyze the role of gyrA mutations in eliciting high quinolone resistance in uropathogenic E.coli (UPEC) through molecular docking studies. Quinolone susceptibility testing of 18 E.coli strains isolated from UTI patients revealed unusually high resistance level to all the quinolones used; especially norfloxacin and ciprofloxacin. The QRDR of gyrA was amplified and sequenced. Mutations identified in gyrA of E.coli included Ser83Leu, Asp87Asn and Ala93Gly/Glu. Contrasting previous reports, we found Ser83Leu substitution in sensitive strains. Strains with S83L, D87N and A93E (A15 and A26) demonstrated norfloxacin MICs ?1024mg/L which could be proof that Asp87Asn is necessary for resistance phenotype. Resistance to levofloxacin was comparatively lower in all the isolates. Docking of 4 quinolones (ciprofloxacin, ofloxacin, levofloxacin and norfloxacin) to normal and mutated E.coli gyrase A protein demonstrated lower binding energies for the latter, with significant displacement of norfloxacin in the mutated GyrA complex and least displacement in case of levofloxacin.
Project description:Fluoroquinolones target two bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. Acquired resistance to quinolones occurs stepwise, with the first mutation occurring in the more sensitive target enzyme. To limit the emergence of resistance, quinolones should ideally possess dual activities against the two enzymes. For reasons that are as yet unclear, Staphylococcus aureus gyrase is less sensitive to quinolones than topoisomerase IV, counter to its greater sensitivity in Escherichia coli, thereby limiting the use of quinolones for the treatment of staphylococcal infections. Mutations in the alpha4-helix domain of the GyrA subunit of gyrase are important in determining quinolone resistance. We replaced an extended region encompassing the alpha4 domain in the E. coli GyrA protein with its homolog in S. aureus and tested for its ability to complement a thermosensitive gyrase and its catalytic and noncatalytic properties. Purified gyrase reconstituted with chimeric GyrA was more resistant to ciprofloxacin than wild-type gyrase at both inhibition of catalytic activity and stimulation of cleavage complexes, and this difference was more apparent in the presence of K+-glutamate. The chimeric GyrA subunit was able to complement thermosensitive gyrase, similar to wild-type GyrA. Without supplemental K+-glutamate the MICs of ciprofloxacin for thermosensitive E. coli complemented with chimeric DNA gyrase were equal to those for E. coli complemented with wild-type gyrase but were twofold higher in the presence of K+-glutamate. Our findings suggest that the extended alpha4 domain of S. aureus GyrA is responsible, at least in part, for the increased resistance of S. aureus gyrase to quinolones and that this effect is modulated by K+-glutamate.
Project description:Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.